Internal variability of rDNA in an ongoing speciation of the yeast Ogatea uvarum Sp.Nov.

A yeast strain was isolated during a large-scale survey yeast strains from vineyard-associated in Apulia (Southern Italy). The sequences of the ITS and LSU D1/D2 domain of the ribosomal RNA (rDNA), revealed that this strain did not fit in to any known species and was consequently described as the type strain of Ogataea uvarum sp.nov. The novel species resulted to be a close relative of O. philodendri. The sequences of the two rDNA domains showed several secondary peaks, indicating internal heterogeneity among the copies of the rDNA operon. This supposition was verified by cloning the region across the ITS1 to LSU D1/D2 and sequencing both strands of each clones. The analyses on the obtained data indicated different levels of variability throughout the operon with differences between the 5.8S and D1/D2 domain of the 26S and the internally transcribed regions (ITS). O. uvarum and O. philodendri share high frequency variants, i.e. variants frequently found in many clones, whereas great variability of the low frequency polymorphisms was found, thus suggesting that the homogenization mechanism is more active with the former than with the latter type of variation. These results indicate that low frequency variants are present in Sanger sequencing as secondary peaks and represent a sort of noise, whereas Next Generation Sequencing (NGS) approach will trace also the secondary variants, with the risk of overestimating the alpha diversity. The significance and the impact of the above finding on the use of these taxonomic markers to investigate the separation of fungal species will be discussed. This research was partially supported by the Apulian Region Projects "Biotecnologie degli alimenti per l'innovazione e la competitività delle principali filiere regionali: estensione della conservabilità e aspetti funzionali - BIOTECA -QCBRAJ6."